Chemopreventive properties of chlorophylls towards aflatoxin B1: a review of the antimutagenicity and anticarcinogenicity data in rainbow trout

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Abstract

The anticarcinogenic activity of chlorophyllin (CHL), a water-soluble derivative of chlorophyll, was first reported in rainbow trout. This review describes certain experiments which set the stage for long-term tumor bioassays, in trout and other species, using CHL and various food-borne carcinogens. Initial work with trout and rat liver enzymes in the Salmonella assay showed that CHL was a potent antimutagen towards heterocyclic amines, polycyclic aromatic hydrocarbons, aflatoxins and other classes of mutagen. Antimutagenic activity was further demonstrated using the corresponding direct-acting mutagens in the absence of an exogenous metabolizing system. Mutagen-inhibitor interaction (molecular complex formation) was identified in spectrophotometry studies, suggesting that CHL acts as an `interceptor molecule'. In vivo, CHL reduced hepatic AFB1-DNA adducts and hepatocarcinogenesis when the inhibitor and carcinogen were co-administered in the diet. Finally, co-injection of inhibitor and AFB1 into trout embryos established that CHL was more effective than chlorophyll a in reducing AFB1-DNA adducts 2 weeks after injection, and liver tumors after 1 year.

Introduction

Waters et al. [1]recently reviewed an extensive database on the activities of several antimutagens in vitro and in vivo, and summarized the results in the form of detailed `activity profiles'. Among the various inhibitors reviewed, chlorophyllin (CHL) was identified as almost uniformly protective against a broad range of direct- and indirect-acting mutagens, including aflatoxins, polycyclic aromatic hydrocarbons, heterocyclic amines, alkylating agents and several miscellaneous compounds. Close inspection of the data presented in the review indicates that the vast majority of experiments using CHL were from assays conducted in vitro, and in many cases these were descriptive rather than mechanistic studies. The antimutagenicity profile for CHL contained only 7 out of a total of 93 data points that were from experiments conducted in vivo. The limited nature of the in vivo data notwithstanding, most of the evidence strongly supported a protective role for CHL [1].

In the time since the review by Waters et al. [1]was submitted and published, several new in vivo studies with CHL have been reported. Perhaps most notable among these is the work that showed CHL to be a potent inhibitor of aflatoxin B1 (AFB1)-initiated tumorigenesis in rainbow trout [2]. This was the first evidence that CHL could operate not only as an antimutagen, but also as an anticarcinogen in some experimental protocols. The present paper reviews several of the key experiments with CHL and AFB1 in trout. In the context of this Special Issue on `The Use of Fish and Fish Transgenics in Genotoxicology Studies', the review serves to illustrate several of the advantages and limitations of rainbow trout as a model for investigating modulators of tumorigenesis and genotoxicity in vivo.

Section snippets

In vitro antimutagenic activity of CHL vs. AFB1 and AFB1-8,9-epoxide

The first studies with CHL in our laboratory used the Salmonella assay, and were based on preliminary experiments which examined the antimutagenic activity of CHL vs. AFB1 [3]. A key observation in the studies by Whong et al. [3]was that the inhibitory activity occurred only when CHL and AFB1 were incubated concurrently in the assay, suggesting that direct interaction between the inhibitor and mutagen might be important for antimutagenic activity. We observed the antimutagenic activity of CHL

Inhibition of AFB1-DNA binding in vivo

Previous work in our laboratory with AFB1 and the anticarcinogen indole-3-carbinol established that AFB1-DNA adducts increased linearly with time of carcinogen exposure during a 2-week dietary treatment of trout, and that the slopes of the DNA binding curves decreased with concentration of inhibitor in the diet [5]. Similar results were obtained in studies with CHL, in which trout were co-treated with AFB1 and the inhibitor for 7 days (Fig. 1b). In these experiments, CHL protected against AFB1

Inhibition of AFB1-DNA adducts and hepatocarcinogenesis (molecular dosimetry studies)

Rainbow trout (1–2 g body weight) were exposed in the diet to 0, 500, 2000, or 4000 ppm CHL and, within each inhibitor group, to one of four different doses of AFB1. The carcinogen was administered in tritiated form during a 2 week period and 15 fish were selected at random from each tank in order to determine AFB1-DNA binding levels in the liver. The remaining animals were switched to control diet and after 9 months 100 of the fish in each tank were selected at random in order to determine the

Interceptor molecule hypothesis

Hartman and Shankel [7]reviewed the literature for inhibitors that interact directly with mutagens and carcinogens and serve in effect to `sequester' these compounds from harm's way. By reducing the bioavailability of many genotoxins, these putative `interceptor molecules' might represent an important first line of defense, perhaps rivaling such mechanisms as induction of detoxification enzymes or inhibition of carcinogen activating enzymes. In the period since the review by Hartman and Shankel

AFB1-CHL complex formation

Evidence for the formation of an AFB1-CHL complex was first obtained in titration studies using a double-beam spectrophotometer. With the addition of AFB1 to the sample cuvette followed by subsequent additions of CHL to both cuvettes, quenching of the absorption spectrum of AFB1 occurred in a manner consistent with complex formation between the two molecules (Fig. 2). Because both compounds have absorption maxima in the region 200–400 nm, which might cause interference during spectrophotometric

Activity of natural chlorophylls in vitro and in vivo

Computer-modeling studies indicate that the tetrapyrrole macrocyclic is the most important feature for complex formation with AFB1, and that natural chlorophylls should be effective inhibitors. However, in contrast to CHL, which is highly water soluble, lipophilic natural chlorophylls complex less effectively with carcinogens under the aqueous conditions of many in vitro assays. A mixture of chlorophylls a and b was shown to exhibit antimutagenic activity in the Salmonella assay by a mechanism

Acknowledgements

The work reviewed here was supported in part by the following Public Health Service grants: CA65525, CA34732, ES03850 and ES00210.

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